TMRE staining to detect mitochondrial membrane potential.

Subconfluent HeLa cells in six-well plates were transfected with 1,000 ng of either pCI, pM1L-V5, or pF1L-FLAG. At 24 h posttransfection, cells were incubated in medium either lacking or containing 5 μM STS (Selleck Chemicals) at 37°C. After 2 h, medium was removed and cells were incubated with fresh medium containing 0.2 μM tetramethylrhodamine ethyl ester (TMRE; Thermo Fisher) for 30 min at 37°C. Cells were harvested by trypsinization, collected by centrifugation (500 × g, 5 min), resuspended in PBS containing 2% FBS, and incubated on ice. Next, samples were analyzed by using a BD FACSCanto II (BD Biosciences) equipped with 488-nm and 633-nm lasers. TMRE dye was excited by the 488-nm laser, and its fluorescence was captured using the 585/42 nm bandpass filter. At least 10,000 events (live and apoptotic cells) were collected for each sample. Cell doublets and debris, identified on the basis of their scatter characteristics, were excluded from analysis. Data analysis was performed using FCS Express 6 (De Novo Software). The percent apoptosis was calculated as the percentage of cells that had low fluorescence divided by the total number of cells. A separate portion of each sample was instead lysed in 100 μl RIPA buffer (150 mM NaCl, 1% sodium deoxycholate, 1% NP-40, 0.1% SDS, and 0.01 M Tris-HCl) containing Halt protease inhibitor (Thermo Scientific) for 20 min at 4°C. Cellular lysates were then centrifuged (18,000 × g, 10 min), clarified supernatants were collected, and 30 μg of protein was used to assess protein expression levels via immunoblotting.